key: cord-0936447-8nxsrod5
authors: Marquez, Alonso; Wysocki, Jan; Pandit, Jay; Batlle, Daniel
title: An update on ACE2 amplification and its therapeutic potential
date: 2020-05-29
journal: Acta Physiol (Oxf)
DOI: 10.1111/apha.13513
sha: 12c48c34cf3fee575467872d174164a8c19e6130
doc_id: 936447
cord_uid: 8nxsrod5

The renin angiotensin system (RAS) plays an important role in the pathogenesis of variety of diseases. Targeting the formation and action of angiotensin II (Ang II), the main RAS peptide, has been the key therapeutic target for last three decades. ACE‐related carboxypeptidase (ACE2), a monocarboxypeptidase that had been discovered 20 years ago, is one of the catalytically most potent enzymes known to degrade Ang II to Ang‐(1‐7), a peptide that is increasingly accepted to have organ ‐protective properties that oppose and counterbalance those of Ang II. In addition to its role as a RAS enzyme ACE2 is the main receptor for SARS‐CoV‐2. In this review, we discuss various strategies that have been used to achieve amplification of ACE2 activity including the potential therapeutic potential of soluble recombinant ACE2 protein and novel shorter ACE2 variants.

The renin-angiotensin system (RAS) in its traditional view entails an enzymatic cascade of reactions leading to the generation of angiotensin II (Ang II), the main peptide of the RAS, which has a variety of biological effects [1] [2] [3] [4] [5] [6] [7] [8] [9] . Angiotensin II is a potent vasoconstrictor and also promotes renal sodium retention, actions that sustain blood pressure and are part of the stress response triggered to maintain circulating volume when survival is threatened by bleeding and other hypovolemic situations [7] [8] [9] . In addition, Ang II has hemodynamic actions that are key to maintain the renal circulation. This peptide, however, exerts a myriad of actions at the tissue level that can be deleterious particularly when sustained chronically [10] [11] [12] . Such adverse actions include pro-inflammatory, pro-proliferative and pro-atherosclerotic effects that are independent of its effect on systemic blood pressure and renal hemodynamic actions [13] [14] [15] [16] [17] . Angiotensin II also increases the production of reactive oxygen species (ROS).

This action results from activation of nicotinamide adenine dinucleotide phosphate (NADPH).

The increase in ROS contributes to the unwanted effects of this peptide 18, 19 .

RAS blockade based on inhibiting the formation of Ang II with ACE inhibitors or blocking the activation of the Ang II type 1 (AT1) receptor is a widely used therapy for kidney and cardiovascular disease. Pathways that regulate the degradation of Ang II may also be important for determining levels of Ang II, particularly at the tissue level 20 . Until recently, however, little attention had been paid to enhancing Ang II degradation as a way to counteract RAS over-activity which is usually present in kidneys from experimental models of diabetic kidney disease and likely in patients with many causes of CKD. Several enzymes are involved in the degradation of Ang II (Figure 1) .

In this review we will focus on an ACE-related carboxypeptidase (ACE2), a homolog of ACE, described in 2000 21, 22 . ACE2 shares 42% homology with the metalloprotease catalytic domains of ACE 22 . ACE2, unlike ACE, contains only one active domain 21, 22 . In its full length form ACE2 has 805 amino acids whereas the soluble form of ACE2 has only 740 amino acids. ACE2 is not inhibited by any of the existing ACE inhibitors. ACE2 acts by

This article is protected by copyright. All rights reserved removing single amino acids from the C terminus of its peptide substrates 23, 24 . It is one of the more catalytically potent enzymes known to convert the vasoconstrictor Ang II to Ang-(1-7) 21,23,25 Ang 1-7 is increasingly accepted to have vascular-protective and reno-protective properties that oppose and counterbalance those of Ang II, such as vasodilation and oxidative stress [26] [27] [28] [29] . Within the renin angiotensin system, the other known target peptide for ACE2 cleavage is Ang I with the subsequent formation of Ang-(1-9) [22] [23] [24] 30 . Recent interest in ACE2 has increased dramatically as a result of the recognition that it is the main receptor for SARS-CoV2, the coronavirus responsible for the current COVID19 pandemic.

Studies in experimental models of either genetically or pharmacologically-induced ACE2 ablation have generally reported deleterious effects in various organs. Therefore, it is not surprising that over the past several years approaches aimed at augmentation of ACE2 activity had gained a significant interest for their therapeutic potential in a variety of pathological conditions. In this review, we will focus on presenting research done in our laboratory and others using various strategies to achieve amplification of ACE2 activity and discuss its therapeutic implications.

Two studies in 2000 reported the existence of a new enzyme that was termed ACE2 21, 22 .

This discovery now 20 years ago created an interest in the ACE2/Ang-(1-7) axis. In general, ACE2 and Ang (1-7) are felt to exert beneficial actions that are organ protective 21, 22, 27 . ACE2 and two other peptidases, prolylcarboxipeptidase (PRCP) and prolylendopeptidase (POP) are the known enzymes responsible for the formation of Ang 1-7 from Ang II (1) (2) (3) (4) (5) (6) (7) (8) . [31] [32] [33] [34] These three enzymes cleave the phenylalanine amino acid from the C-terminal end of Ang II-(1-8) to form Ang 1-7 (Figure 1) . The relative importance varies from tissue to tissue.

For instance, ACE2 is very critical for this action in the kidney whereas POP is the dominant enzyme in lungs and systemic circulation. 33 Ang I (1-10) can also contribute to the formation of Ang (1-7). The conversion to Ang 1-7 I from ANG I is produced by neprilysin. 28, 35, 36 ACE2 contributes to the formation of Ang (1-7) not only by enhancing the formation Ang (1-7) from

This article is protected by copyright. All rights reserved Ang II (1) (2) (3) (4) (5) (6) (7) (8) but also by increasing the formation of Ang-(1-9) from Ang I. Angiotensin (1) (2) (3) (4) (5) (6) (7) (8) (9) could be then converted to Ang 1-7 by ACE 22,32 , neprilysin 37 or cathepsin A 38 . Interestingly, Ang (1-9) could also contribute to the protective effects since this peptide has been proposed to activate the AT2R 39 (Figure 2) .

While this review is limited to the formation of Ang-(1-7) from Ang II by ACE2 it should be noted that ACE2 hydrolyzes several other peptide substrates. Those include apelins (i.e. apelin-13 and apelin-36), the opioid peptide dynorphin A, the kallikrein-kinin-system peptide des-9 Arg bradykinin, and ghrelin, a growth hormone secretagogue 23 . In addition to the catalytic properties of ACE2 this protein may also exert non-enzymatic functions 40 . Of great interest , the membrane-bound ACE2 is known to be the receptor for the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 41 and recently for SARS-COV2 , the coronavirus responsible for COVID-19 42, 43 . (Figure 2 ) Given the rapid emergence of the Cov-19 pandemic, there is increasing interest in ACE2 and its tissue distribution. This enzyme is only present in small amounts in the circulation but heavily expressed in certain organs such as kidneys and in the intestines 33 . In comparison with these organs the expression of ACE2 in the lungs is low 33 but present in alveolar type II pneumocytes 44 . Unlike the full length ACE2, which is anchored to the plasma membrane, the soluble form of ACE2 circulates but in very small amounts. 25 

This article is protected by copyright. All rights reserved 2). Most of the work so far has been done in experimental animals in humans healthy volunteers recombinant ACE2 reduced the level of Ang II. 48 Even though Ang (1-7) infusion failed to demonstrate vascular effects in humans. 49 Sasaki et al reported that Ang (1-7)

infusion increased forearm blood flow in healthy subjects whereas in patients with hypertension there was a minimal effect. In human adipose and atrial micro vessels, Ang (1-7) induces vasodilation via nitric oxide (NO)-dependent and telomerase-dependent processes through MasR, effects that seems to be absent in patients with coronary artery disease. 50 Finally, ACE2/Ang 1-7/Mas receptor axis undoubtedly seems to have a protective effect on different organ and systems. Interestingly different studies, however, reported that Ang (1-7)

has deleterious effects to increase blood pressure and exacerbate cardiac fibrosis in subtotal nephrectomy rats kidney disease model in association with increased cardiac ACE activity 51,52

Several approaches can be used experimentally to increase ACE2 activity ( 

Viral delivery systems using adenovirus, adeno-associated virus or lentivirus, have been used as proof-of-concept approaches to augment ACE2 expression in vivo at the central nervous system and variety of peripheral tissues.

Huentelman et al 53 first used lentiviral vector encoding mouse ACE2 (lenti-mACE2) to amplify ACE2 activity in the heart. Lenti-mACE2 was injected intracardiac in 5-day-old Sprague-Dawley rats. Angiotensin II administration for 4 weeks to control rats resulted in the expected increase in systolic blood pressure, increased weight to body weight ratio and increased myocardial fibrosis. 54 Transduction with lenti-mACE2 resulted in a decrease in the heart weight to body weight ratio and a reduction in the myocardial fibrosis caused by infusion of angiotensin II . 54 This improvement in cardiac hypertrophy was associated with increased expression of ACE2 in cardiac tissue. Similar to Ang II-induced hypertension, in the spontaneously hypertensive rat (SHR) murine ACE2 gene transfer into the heart using lentiviral transduction attenuated hypertension and the associated pathological changes, such as left ventricular wall thickness and of perivascular fibrosis 54 . In addition, an improvement of cardiac function, as evidenced by an increase in left ventricular end diastolic and end systolic diameters in SHR rats after ACE2 overexpression was observed 54 .

The use of lentivirus and adeno-associated viral systems also allowed to generate ACE2 overexpressing transgenic animals for studies examining effects of cardiac-specific ACE2 amplification in adult animals. These viral systems, similar to conditional transgenic models, have the advantage of long-term in vivo ACE2 over-expression that could be "turned on" in adult life and therefore avoid the interference from the effect of developmental ACE2

overexpression. In this respect it should be noted that initial study in traditional transgenic mice with cardiac overexpression of human full-length ACE2 was not encouraging 55 . Even though transgenic mice appeared healthy, they died prematurely. Their diminished survival rates correlated with the extent of ACE2 overexpression in two transgenic lines suggesting a transgene dose effect 55 . Hearts from both transgenics, however, were essentially normal,

This article is protected by copyright. All rights reserved without hypertrophy and similar to wild type. In addition, transgenic and non-transgenic littermates were similar to each other by echocardiography. Moreover, by cardiac catheterization ventricular performance, was similar 55 . The mortality of the transgenic mice could ultimately be explained by electrophysiological analyses that revealed conduction disturbances and lethal ventricular arrhythmias in hACE2 transgenic mice. It still remains to be examined whether or not the discrepancy between the cardiac effects by virally-induced ACE2 overexpression in adult animals and those reported in ACE2 transgenic mice are due to developmental issues.

Yamazato M et al 56 showed the long terms effect of ACE2 on blood pressure. In this study, SHR rats had a relative deficiency in ACE2 protein expression within the cardiovascular regulatory neurons of the rostral ventrolateral medulla (RVLM) when compared with normotensive Wistar-Kyoto rats. Attempted correction of this deficiency in the SHR rats by ACE2 overexpression by lentivirus injection into RVLM resulted in long-term reduction in blood pressure 56 .Therefore central ACE2 overexpression could correct its intrinsic decrease in the RVLM and, similarly to the above discussed cardiac ACE2 overexpression 54 , leading to a substantial blood pressure reduction in the SHR rat 56 .

In lungs, the involvement of RAS in the pathogenesis of certain conditions such as pulmonary hypertension (PH) has been inferred from the high abundance of ACE in the pulmonary vasculature 57, 58 High ACE levels likely contribute to excessive generation of Ang 

This article is protected by copyright. All rights reserved PH-induced lung injury suggesting support for ACE2 as a possible target for upregulating strategies for the treatment of this disease 59

It is also important to mention the protective role of ACE2 on the nervous system. Feng et al 60 studied the effects of ACE2 using a transgenic mouse model with high expression of hACE2 protein in the brain. In this study they concluded that over-expression of ACE2 would attenuate the development of neurologic hypertension as a consequence of attenuation of parasympathetic tone and spontaneous baroreflex sensitivity. Of note, neurological deficits improvement and cerebral infarct size reduction after a neurological ischemic event have been attributed antioxidative and anti-inflammatory effects of ACE2/Ang (1-7)/Mas axis. 61 Therefore, ACE2 could also be a target to prevent and treat ischemic stroke in the future.

Various studies have shown a role of ACE2 in kidney disease. Nadarajah et al. generated a model of glomerular ACE2 overexpression using a podocyte-specific ACE2 transgenic mice and showed partial protection against the early development of albuminuria 62 . Preservation of podocyte proteins and podocyte number, were seen in STZ-induced diabetes transgenic mice with overexpression of the human ACE2 protein 62 . That kidney over-expressed ACE2

can ameliorate glomerular injury in diabetic animals was also suggested by a study using adenoviral kidney ACE2 (Ad-ACE2) overexpression in STZ rats 63 . Compared with control, the Ad-ACE2 treated group showed a reduction in systolic blood pressure and improvement in urinary albumin excretion, creatinine clearance and glomeruli sclerosis index. Ad-ACE2 also had decreased TGF-β1, vascular endothelial growth factor and collagen IV protein expression. 63 No additional benefit of ACE inhibition was noticed in the combined use of Ad-ACE2 and ACEI 63 . Overall these studies suggest that kidney ACE2 amplification may represent a therapeutic target in the treatment of glomerular injury in diabetic kidney disease.

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Minicircle DNA vectors consist of a circular expression cassette devoid of the bacterial plasmid DNA backbone that provides sustained transgene expression in quiescent cells/tissues 64 . We studied the effects of murine recombinant ACE2 in streptozotocininduced diabetes in mice as well as the effect of increasing circulating ACE2 using minicircle DNA delivery. 65 This approach resulted in sustained increase in serum ACE2 activity and enhanced ability to degrade infused angiotensin II (1) (2) (3) (4) (5) (6) (7) (8) . 64 . In mice with streptozotocininduced diabetes pretreated with ACE2, minicircles, plasma ACE2 protein increased as shown by western blot and ACE2 serum activity increased more than 100-fold. 65 Urinary ACE2 activity and kidney ACE2, however, did not Increase despite the profound augmentation of ACE2 in plasma. Moreover, the glomerular lesions and hyperfiltration seen in this diabetic model of experimental kidney disease were not affected at all l 65 . From these findings we concluded that, targeting kidney ACE2, rather than circulating plasma ACE2, might be necessary to effectively treat early diabetic kidney disease as seen in the STZ-model.

ACE2 in its full-length form is a 110-120kDa-protein comprising 805 amino-acids (aa). It is a type-I transmembrane protein that contains a major extracellular domain (aa 1-740), and the much smaller: transmembrane region (aa 741-768), and intracellular tail (769-805) 66 

This article is protected by copyright. All rights reserved of acute Ang II dependent hypertension. (Figure 3 ). Imai et al. 68 used hrACE2 to examine its effect on experimental acute lung injury. Using aspiration-induced acute lung injury murine model they injected human recombinant ACE2 (hrACE2) into acid-treated Ace2 knockout mice and observed a decrease in the degree of acute lung injury, and pulmonary edema formation. hrACE2 has also been proposed as a potential candidate to treat diastolic and systolic heart failure 69 . Zhong et al 70 showed that hrACE2 ameliorated pressure overload induced as well as Ang II-induced myocardial remodeling. hrACE2 has also shown to reduce the level of Angiotensin II in healthy human volunteers. Despite the ACE blockade the levels of Angiotensin II could remain elevated via chymase system. 48 All of the above suggest that therapies using hrACE2 for heart failure seem promising.

Human recombinant ACE2 has been evaluated in human subjects in limited clinical studies .The pharmacokinetics, pharmacodynamics, safety, and tolerability of hrACE2 were determined in healthy volunteers 71 . This study was randomized, double-blind, and placebo- 

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Encouraging but still very preliminary findings were drawn from a recent phase IIa, openlabel pilot study which suggested a potential therapeutic role for hrACE2 in pulmonary arterial hypertension 74 . This study found a reduced plasma ACE2 activity in subjects with PH. This was inferred, from higher plasma Ang II to Ang (1-7) ratio however, the ratio is not specific as it can potentially be affected by changes in other enzymes that affect the conversion of Ang II to Ang (1-7) . At baseline in subjects with PH, increased expression in six out of nine measured cytokines as compared with controls (interleukin (IL)-10, IL-1β, tumor necrosis factor (TNF)-α, IL-13, IL-8 and IL-4) was found. Reduced plasma superoxide dismutase 2 (SOD2), which is considered an anti-oxidant enzyme, and increased oxidant stress parameters were also observed 74 . After hrACE2, cytokines such as IL-10, IL-1β, IL-2

and TNF-α were decreased 2 hours after administration. Human rACE2 administration was reported to also beneficially influence SOD2 levels, and reduce plasma oxidant stress.

These findings were based on a limited number of subjects. Further assessment of hrACE2 as a potential therapeutic in PH certainly will require larger studies. Table 2 provides a summary of studies so far that have used human soluble recombinant ACE2.

While the human rACE2 is the ultimate form of the protein that can be used in clinical studies, it has some limitations for preclinical research in rodents. Human rACE2 can certainly be used in acute studies in rodents. There is limited value, however, for chronic studies due to the development of antibodies that decrease the enzymatic activity of ACE2 

This article is protected by copyright. All rights reserved rACE2 of 1-740 AA might not be effective. Consistent with this notion, we have found that in STZ-treated mice, a diabetic mouse model with local kidney but not systemic RAS overactivity 65 , long-term augmentation of circulating ACE2 activity was not sufficient to beneficially alter albuminuria, GFR or kidney histology. 65 Glomerular filtration of administered rACE2 is a prerequisite for a subsequent tubular uptake of the protein from the urinary space 65, 80 . Kidney targeting of biologics is complex and can be affected by many factors including their molecular size 81 . We reasoned that in forms of kidney disease with an overactive RAS within the kidney 65,82 ACE2 amplification would require forms of recombinant ACE2 that are short enough to be able to pass the glomerular filtration barrier and be consequently reabsorbable by the kidney to be able to exert any direct kidneyspecific therapeutic effect. Based on these considerations, we have generated ACE2

variants of shorter molecular size that still retain enzymatic activity (see below).

We have generated and tested two novel recombinant mouse ACE2 proteins of a molecular size (~69-71kD) which are much shorter than the original soluble rACE2 of 100- 

This article is protected by copyright. All rights reserved longer than that of ACE2 

This article is protected by copyright. All rights reserved soluble rACE2. In addition, ex vivo kidney cortex lysates from ACE2 1-619-injected mice were able to form significantly more Ang (1-7) from Ang II than kidney lysates from PBS-or original soluble rACE2-injected mice). In the aggregate these data demonstrate that short ACE2 variants are active, and sufficiently small to be filtered by the kidney and moreover capable to increase kidney ACE2 activity to an extent that Ang (1-7) formation from Ang II is increased.

The crystal structure of ACE2 was solved by Hernández Prada et al 85 and 3 putative small molecule binding pockets were identified. These authors carried out in silico screening of a small molecule library followed by in vitro studies and found that out of the 3 sites, two of them were inhibitor sites and the third one was a presumed activator site. The same investigators 85 identified then two small-molecular compounds as ACE2 activators: XNT and resorcinolnaphthalein. XNT was preferred over resorcinolnaphthalein since XNT solubility properties appeared to be more favorable. 85 Three years later an agent commonly used in to treat some parasitizes in animals 86 called Diminazene (DIZE), was proposed as an ACE2 activator 87 . Both XNT and DIZE have been used experimentally to serve as potential treatment for various conditions such as certain types of hypertension 85-88 , pulmonary hypertension [89] [90] [91] . Cardiac and renal fibrosis 85 and glaucoma 92 .The therapeutic benefits of these two components should be attributable to conversion of Ang II to Ang (1-7) as a consequence of ACE2 activation 86, 87, [89] [90] [91] [92] . One of the caveats with many of these studies is, however, that the effect on ACE2 activity was usually not reported in vivo 85,87,89-91,93-100 . In these studies moreover it was not shown that the use of these presumed ACE2 activators had taken place by demonstrating the enhanced conversion of Ang II to Ang (1-7).

Our group reported low levels plasma ACE2 activity in both vehicle and XNT infused mice 65,80 . Furthermore, after Ang II infusion the plasma levels Ang (1-7) , the peptide generated by the cleavage of Ang II and plasma levels Ang II, the substrate of ACE2, were not affected by the administration of XNT. Therefore, it was suggested that the effect of XNT on Ang II-

This article is protected by copyright. All rights reserved in a cell free system and also to upregulate ace2 mRNA expression in cultured cells 103 .

There have been no follow up studies, to our knowledge, with the IRW compound which would independently assess its potency as an ACE2 activator. As of today there is no convincing evidence, in our opinion, that the presumed ACE2 activators studied exert their otherwise undisputable biologic effects by activating ace2 as their main mechanism of action

From the forgoing, it is logical to postulate that ACE2 amplification within the kidney is a very attractive therapeutic approach to promote the metabolism of Ang II and reduce its detrimental actions. Some of the potential diseases that could benefit from ACE2 amplification have been mentioned already while discussing the methods up to attempt to increase ACE2 activity. As noted earlier, partial kidney protection against STZ-induced diabetic kidney disease could be shown in a transgenic model where ACE2 overexpression was limited to glomerular podocytes 62 . In one study human recombinant ACE2 was shown to improve diabetic kidney disease in Akita mice 104 . When these mice were injected with hrACE2 for 4 weeks, hrACE2 normalized blood pressure and reduced albuminuria. Unlike

other models of early DKD the Akita mouse is hypertensive and is very possible that the improvement in albuminuria noted in this study was related to lowering of blood pressure by ACE2 by decreasing Ang II which is elevated in this model.

In keeping with previous studies in mice by Ye et al. 105 in ACE2 activity as well as an enhanced ability to metabolize acute load of Ang II resulted from ACE2 minicircle delivery However, this augmented ACE2 activity achieved by this technique did not affect urine ACE2 activity. Moreover, there were no improvements in albuminuria, glomerular expansion, glomerular cellularity or glomerular size when compared to vehicle -treated diabetic controls. (Figure 4 ) Therefore, this study emphasized the importance of targeting the kidney rather the circulating RAS in order to treat diabetic nephropathy.

In other study using Col4a3 -/-mouse model of Alport's syndrome, murine recombinant (mr)

ACE2 also decreased markers of kidney injury. ACE2 plasma activity was increased using mrACE2 mini-pumps leading to an improvement in albuminuria, reduced ERK1/ERK2

This article is protected by copyright. All rights reserved signaling and amelioration of inflammation, fibrosis and oxidative stress 108 . In this study renal function did not improve as measured by blood urea nitrogen and GFR was not measured. In this model the profound defect in glomerular permeability allows the infused rACE2, despite its large molecular size, to get filtered 65 [110] [111] [112] [113] [114] , but also in AKI and are associated with adverse outcomes both in experimental and clinical studies [115] [116] [117] [118] [119] [120] [121] [122] . We think that AKI could be an initial target to explore in humans the potential preventative effect of our short ACE2 variants.

Finally, this review comes at a time that the world is grappling with the COVID19 pandemic caused by SARS-CoV2 virus. From the SARS outbreak in 2003 we have known that SARS-CoV1 spike glycoprotein recognized ACE2 as a receptor on the cell surface for host entry 41 .Consequently, there have already been several reports looking at the association of SARS-CoV2 and ACE2 43, 123 . It is known that SARS-CoV2 does bind to ACE2 to gain host cell entry causing its cell internalization and likely reducing membrane ACE2 expression 124 In murine models the dysregulation of ACE2 is associated with cardiac, pulmonary and kidney alterations . There is preliminary data from COVID-19 subjects in whom elevated levels of plasma angiotensin II correlated with degree of lung injury 125 .

Further prior preclinical studies in respiratory syncytial virus and avian H5N1 influenza

This article is protected by copyright. All rights reserved suggested that restoration of ACE2 by recombinant ACE2 administration appeared to reverse worsening lung injury 73, 126, 127 . Therefore, there is now significant interest in looking at recombinant ACE2 protein to rebalance the RAS network and potentially help mitigate the pulmonary, cardiac and kidney damage done by COVID 19. We have proposed that soluble ACE2 may act as a competitive interceptor of SARS-CoV and SARS-CoV2 by preventing binding of the viral particle to the surface-bound, full-length ACE2. 45 .In this context, administration of soluble recombinant human ACE2 proteins might be beneficial as a novel biologic therapeutic to limit or even combat the infection progression caused by coronaviruses that utilize ACE2 as a receptor. 

Regulation of blood pressure by the type 1A angiotensin II receptor gene

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The Renin-Angiotensin System and the Neurodegenerative Diseases: A Brief Review

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American journal of kidney diseases : the official journal of the National Kidney Foundation

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Accepted Article 34

Update on the Angiotensin converting enzyme 2-Angiotensin (1-7)-MAS receptor axis: fetal programing, sex differences, and intracellular pathways

In vivo metabolism of angiotensin I by neutral endopeptidase (EC 3.4.24.11) in spontaneously hypertensive rats

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Accepted Article This article is protected by copyright. All rights reserved 45

Soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy

Tissue-specific amino acid transporter partners ACE2 and collectrin differentially interact with hartnup mutations

Angiotensin converting enzyme (ACE) and ACE2 bind integrins and ACE2 regulates integrin signalling

Role of the ACE2/Angiotensin 1-7 Axis of the Renin-Angiotensin System in Heart Failure

Angiotensin-(1-7) does not affect vasodilator or TPA responses to bradykinin in human forearm

Vascular Actions of Angiotensin 1-7 in the Human Microcirculation: Novel Role for Telomerase

Adverse cardiac effects of exogenous angiotensin 1-7 in rats with subtotal nephrectomy are prevented by ACE inhibition

Angiotensin-(1-7) infusion is associated with increased blood pressure and adverse cardiac remodelling in rats with subtotal nephrectomy

Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats

ACE2 gene transfer attenuates hypertension-linked pathophysiological changes in the SHR

Accepted Article This article is protected by copyright. All rights reserved 55

Heart block, ventricular tachycardia, and sudden death in ACE2 transgenic mice with downregulated connexins

Overexpression of angiotensin-converting enzyme 2 in the rostral ventrolateral medulla causes long-term decrease in blood pressure in the spontaneously hypertensive rats

Role of angiotensin-converting enzyme and angiotensin II in development of hypoxic pulmonary hypertension

Expression of pulmonary vascular angiotensin-converting enzyme in primary and secondary plexiform pulmonary hypertension

Prevention of pulmonary hypertension by Angiotensin-converting enzyme 2 gene transfer

Brain-selective overexpression of human Angiotensin-converting enzyme type 2 attenuates neurogenic hypertension

ACE2-Ang-(1-7)-Mas Axis in Brain: A Potential Target for Prevention and Treatment of Ischemic Stroke

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme (ACE) 2 overexpression ameliorates glomerular injury in a rat model of diabetic nephropathy: a comparison with ACE inhibition

A robust system for production of minicircle DNA vectors

Accepted Article This article is protected by copyright. All rights reserved 65

Angiotensin-converting enzyme 2 amplification limited to the circulation does not protect mice from development of diabetic nephropathy

Angiotensin-converting enzyme 2 and angiotensin 1-7: novel therapeutic targets

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

Angiotensin-converting enzyme 2 protects from severe acute lung failure

Recombinant human angiotensin-converting enzyme 2 as a new renin-angiotensin system peptidase for heart failure therapy

Angiotensin-converting enzyme 2 suppresses pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction

Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects

Recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow and oxygenation in lipopolysaccharide-induced lung injury in piglets

A pilot clinical trial of recombinant human angiotensinconverting enzyme 2 in acute respiratory distress syndrome

A potential therapeutic role for angiotensin-converting enzyme 2 in human pulmonary arterial hypertension. The European respiratory journal

Murine recombinant angiotensin-converting enzyme 2: effect on angiotensin II-dependent hypertension and distinctive angiotensin-Accepted Article This article is protected by copyright. All rights reserved converting enzyme 2 inhibitor characteristics on rodent and human angiotensin-converting enzyme 2

Recombinant Expression and Characterization of Human and Murine ACE2: Species-Specific Activation of the Alternative Renin-Angiotensin-System

Regulation of urinary ACE2 in diabetic mice

Angiotensin-converting enzyme 2-independent action of presumed angiotensin-converting enzyme 2 activators: studies in vivo, ex vivo, and in vitro

Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation

Novel Variants of Angiotensin Converting Enzyme-2 of Shorter Molecular Size to Target the

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Characterization of proteinuria and tubular protein uptake in a new model of oral L-lysine administration in rats

Urinary Renin in Patients and Mice With Diabetic Kidney Disease

Accepted Article This article is protected by copyright. All rights reserved 85

Structure-based identification of smallmolecule angiotensin-converting enzyme 2 activators as novel antihypertensive agents

Diminazene aceturate (Berenil) modulates the host cellular and inflammatory responses to Trypanosoma congolense infection

Prediction of off-target effects on angiotensin-converting enzyme 2

Activation of angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis attenuates the cardiac reactivity to acute emotional stress

Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension

Diminazene aceturate improves autonomic modulation in pulmonary hypertension

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2

Diminazene Aceturate Enhances Angiotensin-Converting Enzyme 2 Activity and Attenuates Ischemia-Induced Cardiac Pathophysiology

Angiotensin-converting enzyme 2 activation protects against hypertension-induced cardiac fibrosis involving extracellular signal-regulated kinases

Angiotensin-converting enzyme 2 activation improves endothelial function

Accepted Article This article is protected by copyright. All rights reserved 96

ACE2 activation promotes antithrombotic activity. Molecular medicine

Activation of angiotensin-converting enzyme 2/Angiotensin-(1-7)/Mas axis attenuates the cardiac reactivity to acute emotional stress

Chronic activation of endogenous angiotensin-converting enzyme 2 protects diabetic rats from cardiovascular autonomic dysfunction

Oral administration of an angiotensin-converting enzyme 2 activator ameliorates diabetes-induced cardiac dysfunction. Regulatory peptides

Antiglaucomatous Effects of the Activation of Intrinsic Angiotensin-Converting Enzyme 2

Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology

Diminazene aceturate prevents nephropathy by increasing glomerular ACE2 and AT2 receptor expression in a rat model of type1 diabetes

Egg White-Derived Tripeptide IRW (Ile-Arg-Trp) Is an Activator of Angiotensin Converting Enzyme 2

Human recombinant ACE2 reduces the progression of diabetic nephropathy

Glomerular localization and expression of Angiotensin-converting enzyme 2 and Angiotensin-converting enzyme: implications for albuminuria in diabetes

Accepted Article This article is protected by copyright. All rights reserved

American journal of kidney diseases : the official journal of the National Kidney Foundation

Decreased glomerular and tubular expression of ACE2 in patients with type 2 diabetes and kidney disease

Murine recombinant angiotensin-converting enzyme 2 attenuates kidney injury in experimental Alport syndrome

ACE2 as therapy for glomerular disease: the devil is in the detail

Role of the intrarenal renin-angiotensin-aldosterone system in chronic kidney disease

The intrarenal renin-angiotensin system and diabetic nephropathy

The renin-angiotensin system and diabetic nephropathy

Renal renin-angiotensin system in diabetes: functional, immunohistochemical, and molecular biological correlations

The role of renin-angiotensin-aldosterone system genes in the progression of chronic kidney disease: findings from the Chronic Renal Insufficiency Cohort (CRIC) study

Angiotensinogen as a biomarker of acute kidney injury

Role of AT1 angiotensin II receptors in renal ischemic injury

ACE2-angiotensin-(1-7)-Mas axis in renal ischaemia/reperfusion injury in rats

Differential actions of renal ischemic injury on the intrarenal angiotensin system

Fiend and friend in the renin angiotensin system: An insight on acute kidney injury

Acute kidney injury

Intracardiac and intrarenal renin-angiotensin systems: mechanisms of cardiovascular and renal effects

Intrarenal angiotensin and bradykinin peptide levels in the remnant kidney model of renal insufficiency

Renin-Angiotensin-Aldosterone System Inhibitors in Patients with Covid-19

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury

Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury

Angiotensin-converting enzyme 2 protects from lethal avian influenza A H5N1 infections

Angiotensin-converting enzyme 2 inhibits lung injury induced by respiratory syncytial virus

Accepted Article